The disclosures referred to herein to illustrate the background of the invention and to provide additional detail with respect to its practice are incorporated herein by reference and, for convenience, are numerically referenced in the following text and respectively grouped in the appended bibliography.
Oligonucleotide-directed site-specific mutagenesis is a powerful method that is commonly used to create desired mutations. Three basic methods have been used for this purpose: the use of single-stranded (M13),.sup.1,2 the polymerase chain reaction (PCR),.sup.3-6 and the double-stranded plasmid method..sup.7-9 The single-stranded method originally developed by Zoller and Smith has been used for years..sup.10 Many modifications have been made to achieve a higher yield of mutants. The PCR method also has become another popular technique for site-directed mutagenesis. The advantages and shortcomings of these methods have been discussed..sup.11,12 Briefly, limitations include the availability of restriction sites for subcloning and the instability of large insertions (larger than 2 kilobases) in M13 vectors,.sup.13 the low fidelity of the Taq polymerase and the expense for multiple primers in the PCR methods, and the low mutant yields with the double-stranded plasmid method.